Yeast expressed cytochrome P450 2D6 (CYP2D6) exposed on the external face of plasma membrane is functionally competent

ABSTRACT CYP2D6, a xenobiotic metabolizing cytochrome P450 (P450), was found to be present in significant amount on the outer face of cell plasma membrane in addition to the regular microsomal location. Present work demonstrates that this external P450 is catalytically competent and that activity is supported by NADPH-P450 reductase present on the inner face of plasma membrane. Purified plasma membranes from yeast expressing CYP2D6 sustained NADPH-and cumene hydroperoxide-dependent dextromethorphan demethylation and NADPH-cytochrome c activity confirming previous observations in human hepatocytes. CYP2D6 found on the outside of plasma membrane (by differential immuno-inhibition and acidic shift assays on transformed spheroplasts) was catalytically competent at the cell surface for NADPH-supported activities. Anti-yeast P450-reductase antibodies inhibited neither CYP2D6 nor P450-reductase activities upon incubation with intact spheroplasts. In contrast, both activities were inhibited on isolated plasma membrane fragments. This highly suggested a cytosolic-orientation of the plasma membrane P450-reductase. This finding was confirmed by immunostaining in confocal microscopy. Finally, gene deletion of P450-reductase caused a complete loss of plasma membrane NADPH-supported CYP2D6 activity, which suggests that the reductase participates to some degree in the transmembrane electron transfer chain. This work illustrates that the outside-exposed plasma membrane CYP2D6 is active and may play an important metabolic role

A common antitussive drug, clobutinol, precipitates the long QT syndrome 2

QT prolongation, a classic risk factor for arrhythmias, can result from a mutation in one of the genes governing cardiac repolarization and also can result from the intake of a medication acting as blocker of the cardiac K(+) channel human ether-a-go-go-related gene (HERG). Here, we identified the arrhythmogenic potential of a nonopioid antitussive drug, clobutinol. The deleterious effects of clobutinol were suspected when a young boy, with a diagnosis of congenital long QT syndrome, experienced arrhythmias while being treated with this drug. Using the patch-clamp technique, we showed that clobutinol dose-dependently inhibited the HERG K(+) current with a half-maximum block concentration of 2.9 microM. In the proband, we identified a novel A561P HERG mutation. Two others long QT mutations (A561V and A561T) had been reported previously at the same position. None of the three mutants led to a sizeable current in heterologous expression system. When coexpressed with wild-type (WT) HERG channels, the three Ala561 mutants reduced the trafficking of WT and mutant heteromeric channels, resulting in decreased K(+) current amplitude (dominant-negative effects). In addition, A561P but not A561V and A561T mutants induced a approximately -11 mV shift of the current activation curve and accelerated deactivation, thereby partially counteracting the dominant-negative effects. A561P mutation and clobutinol effects on the human ventricular action potential characteristics were simulated using the Priebe-Beuckelmann model. Our work shows that clobutinol has limited effects on WT action potential but should be classified as a "drug to be avoided by congenital long QT patients" rather than as a "drug with risk of torsades de pointes

Transfer of Rolf S3-S4 Linker to hERG Eliminates Activation Gating but Spares Inactivation

Studies in Shaker, a voltage-dependent potassium channel, suggest a coupling between activation and inactivation. This coupling is controversial in hERG, a fast-inactivating voltage-dependent potassium channel. To address this question, we transferred to hERG the S3-S4 linker of the voltage-independent channel, rolf, to selectively disrupt the activation process. This chimera shows an intact voltage-dependent inactivation process consistent with a weak coupling, if any, between both processes. Kinetic models suggest that the chimera presents only an open and an inactivated states, with identical transition rates as in hERG. The lower sensitivity of the chimera to BeKm-1, a hERG preferential closed-state inhibitor, also suggests that the chimera presents mainly open and inactivated conformations. This chimera allows determining the mechanism of action of hERG blockers, as exemplified by the test on ketoconazole